Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Methodol. Sep 26, 2013; 3(3): 27-38
Published online Sep 26, 2013. doi: 10.5662/wjm.v3.i3.27
Bifunctional staining for ex vivo determination of area at risk in rabbits with reperfused myocardial infarction
Yuanbo Feng, Zhan-Long Ma, Feng Chen, Jie Yu, Marlein Miranda Cona, Yi Xie, Yue Li, Yicheng Ni
Yuanbo Feng, Feng Chen, Jie Yu, Marlein Miranda Cona, Yicheng Ni, Theragnostic Laboratory, Department of Imaging and Pathology, Biomedical Sciences Group, 3000 KU Leuven, Belgium
Zhan-Long Ma, Departments of Radiology, the First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Yi Xie, Departments of Electronics and Information System (ELIS), Ghent University, Sint-Pietersnieuwstraat 25, 9000 Gent, Belgium
Yue Li, Lab of Translational Medicine, Jiangsu Academy of Traditional Chinese Medicine, nanjing 210028, Jiangsu Province, China
Yicheng Ni, Radiology Section, University Hospitals, 3000 Leuven, Belgium
Author contributions: All authors made a substantial contribution to the conception and design of the manuscript, drafting the article or revising it.
Supported by The awarded grants of the KU Leuven Molecular Small Animal Imaging Center MoSAIC (KUL EF/05/08); the Center of Excellence in vivo Molecular Imaging Research (IMIR) of KU Leuven; a EU Project Asia-Link CfP 2006-Europe Aid/123738/C/ACT/Multi-Proposal, No. 128-498/111; and Jiangsu Province Natural Science Foundation, China, No. BK2010594
Correspondence to: Yicheng Ni, MD, PhD, Professor, Radiology Section, University Hospitals, KU Leuven Herestraat 49, 3000 Leuven, Belgium. yicheng.ni@med.kuleuven.be
Telephone: +32-16-330165 Fax: +32-16-343765
Received: July 12, 2013
Revised: July 16, 2013
Accepted: August 16, 2013
Published online: September 26, 2013
Processing time: 96 Days and 18.4 Hours
Abstract

AIM: To develop a method for studying myocardial area at risk (AAR) in ischemic heart disease in correlation with cardiac magnetic resonance imaging (cMRI).

METHODS: Nine rabbits were anesthetized, intubated and subjected to occlusion and reperfusion of the left circumflex coronary artery (LCx) to induce myocardial infarction (MI). ECG-triggered cMRI with delayed enhancement was performed at 3.0 T. After euthanasia, the heart was excised with the LCx re-ligated. Bifunctional staining was performed by perfusing the aorta with a homemade red-iodized-oil (RIO) dye. The heart was then agar-embedded for ex vivo magnetic resonance imaging and sliced into 3 mm-sections. The AAR was defined by RIO-staining and digital radiography (DR). The perfusion density rate (PDR) was derived from DR for the AAR and normal myocardium. The MI was measured by in vivo delayed enhancement (iDE) and ex vivo delayed enhancement (eDE) cMRI. The AAR and MI were compared to validate the bifunctional straining for cardiac imaging research. Linear regression with Bland-Altman agreement, one way-ANOVA with Bonferroni’s multiple comparison, and paired t tests were applied for statistics.

RESULTS: All rabbits tolerated well the surgical procedure and subsequent cMRI sessions. The open-chest occlusion and close-chest reperfusion of the LCx, double suture method and bifunctional staining were successfully applied in all animals. The percentage MI volumes globally (n = 6) and by slice (n = 25) were 36.59% ± 13.68% and 32.88% ± 12.38% on iDE, and 35.41% ± 12.25% and 32.40% ± 12.34% on eDE. There were no significant differences for MI determination with excellent linear regression correspondence (rglobal = 0.89; rslice = 0.9) between iDE and eDE. The percentage AAR volumes globally (n = 6) and by slice (n = 25) were 44.82% ± 15.18% and 40.04% ± 13.64% with RIO-staining, and 44.74% ± 15.98% and 40.48% ± 13.26% by DR showing high correlation in linear regression analysis (rglobal = 0.99; rslice = 1.0). The mean differences of the two AAR measurements on Bland-Altman were almost zero, indicating RIO-staining and DR were essentially equivalent or inter-replaceable. The AAR was significantly larger than MI both globally and slice-by-slice (P < 0.01). After correction with the background and the blank heart without bifunctional staining (n = 3), the PDR for the AAR and normal myocardium was 32% ± 15% and 35.5% ± 35%, respectively, which is significantly different (P < 0.001), suggesting that blood perfusion to the AAR probably by collateral circulation was only less than 10% of that in the normal myocardium.

CONCLUSION: The myocardial area at risk in ischemic heart disease could be accurately determined postmortem by this novel bifunctional staining, which may substantially contribute to translational cardiac imaging research.

Keywords: Reperfused; Acute myocardial infarction; Rabbit model; Cardiac magnetic resonance imaging; Oil-red-o dye; Iodized oil

Core tip: For developing therapeutic procedures/drugs aimed at modulating infarct size after coronary artery disease, it is important not only to measure myocardial infarction but also to know the extent of area at risk (AAR). However, determination of the AAR both in vivo and ex vivo can be challenging, and controversial. In this experiment we sought to develop a reliable method to accurately localize the culprit coronary occlusion for postmortem verification after in vivo cardiac magnetic resonance imaging and to establish a new bifunctional staining as a standard reference for ex vivo area at risk identification, which may substantially contribute to translational cardiac imaging research.